Spore Discharge in Basidiomycetes: III. Spore Liberation from Narrow Hymenial Tubes

The hymenial tubes of Polyporus betulinus were shown to be approximatelycylindrical,and to have a sporulating hymenium over the greater part oftheir surface. Sporophores were tilted through measured anglesfrom the vertical in the field and in the laboratory, and theeffect on spore liberation was measured by determining the numberof spores liberated from particular tubes in standard time.All tilting caused a reduction in spore liberation. The amountof this reduction was found to compare closely with that predictedfrom calculation of the amount of hymenium lying directly abovethe orifice at various angles of tilt. It is therefore concludedthat liberation from the tube can be accounted for by the initialviolent discharge of the spore and gravitational attractionalone. An addendum corrects an earlier paper (1959) in thisseries. It shows that the variation in density of spore depositscollected from Trametes gibbosa was due to air currents in theapparatus, not to variation in sporulation rate.     

Adaptation of the Spore Discharge Mechanism in the Basidiomycota

Background

Spore discharge in the majority of the 30,000 described species of Basidiomycota is powered by the rapid motion of a fluid droplet, called Buller''s drop, over the spore surface. In basidiomycete yeasts, and phytopathogenic rusts and smuts, spores are discharged directly into the airflow around the fungal colony. Maximum discharge distances of 1–2 mm have been reported for these fungi. In mushroom-forming species, however, spores are propelled over much shorter ranges. In gilled mushrooms, for example, discharge distances of <0.1 mm ensure that spores do not collide with opposing gill surfaces. The way in which the range of the mechanism is controlled has not been studied previously.

Methodology/Principal Findings

In this study, we report high-speed video analysis of spore discharge in selected basidiomycetes ranging from yeasts to wood-decay fungi with poroid fruiting bodies. Analysis of these video data and mathematical modeling show that discharge distance is determined by both spore size and the size of the Buller''s drop. Furthermore, because the size of Buller''s drop is controlled by spore shape, these experiments suggest that seemingly minor changes in spore morphology exert major effects upon discharge distance.

Conclusions/Significance

This biomechanical analysis of spore discharge mechanisms in mushroom-forming fungi and their relatives is the first of its kind and provides a novel view of the incredible variety of spore morphology that has been catalogued by traditional taxonomists for more than 200 years. Rather than representing non-selected variations in micromorphology, the new experiments show that changes in spore architecture have adaptive significance because they control the distance that the spores are shot through air. For this reason, evolutionary modifications to fruiting body architecture, including changes in gill separation and tube diameter in mushrooms, must be tightly linked to alterations in spore morphology.     

Goldilocks mushrooms: How ballistospory has shaped basidiomycete evolution

Ballistospory has been a governing factor in mushroom diversification. Modifications to fruit body morphology are subject to a series of fundamental constraints imposed by this uniquely fungal mechanism. Gill spacing in lamellate mushrooms, tube width in poroid species, and other configurations of the hymenium must comply with the distance that spores shoot themselves from their basidia. This reciprocal relationship between the development of fruit bodies and spores may have been maintained by a form of evolutionary seesaw proposed in this article. The necessity of the accurate gravitropic orientation of gills and tubes is another constraint on mushroom development and physiology, along with the importance of evaporative cooling of the hymenium for successful spore discharge and the aerodynamic shaping of the fruit body to aid dispersal. Ballistospory has been lost in secotioid and gasteroid basidiomycetes whose spores are dispersed by animal vectors and has been replaced by alterative mechanisms of active spore discharge in some species. Partnered with the conclusions drawn from molecular phylogenetic research, the biomechanical themes discussed in this review afford new ways to think about the evolution of basidiomycetes.     

Spore discharge in Entomophthora grylli

Entomophthora grylli Fres. has been found on larvae of Bradysia (Sciaridae, Diptera) on wood in Kansas State University greenhouses since November 1967. Infected larvae crawled to an exposed site in the night and by morning spores of E. grylli were being discharged. In the greenhouse and under controlled environments spore discharge showed a marked periodicity; spore discharge occurred in the light with peaks in the first 1–4 h; discharge then gradually declined but extended into the dark. On the second and third days peaks occurred in the light but were progressively smaller. In a greenhouse under fluctuating conditions twin peaks occurred at 10·00 and 22·00; in a growth chamber, at constant 21° C. 90% r.h., alternating 12 h light and dark periods, spore discharge was similar, with maximum numbers about 21/2 h after the onset of the photo-period. In continuous dark and continuous light no endogenous pattern was evident. Temperature range for spore discharge was 2–28·, with optimum at 15·. Decreasing humidities resulted in decreased spore production and cessation of discharge below 40% r.h.     

Periodicity of Spore Discharge in Daldinia

In Daldinia concentrica spore discharge under natural conditionsis periodic, most spores being discharged during the night andfew in the daytime. An experimental study has been made of dischargeunder controlled conditions of light and temperature. In continuousdarkness periodic discharge was maintained for 12 days, butthen, although spore output continued, it ceased to be periodic.Return to alternating light (12 hrs., 100 f.c.) and darkness(12 hrs.) at once re-established the periodicity. In continuouslight (100 f.c.) periodic discharge ceased after 2 to 3 days,but was immediately re-established in alternating light (12hrs.) and darkness (12 hrs.). When the fungus was placed underconditions of alternating light and darkness each of 6 hours'duration, two peaks of spore-output were soon developed in the24-hour period. The experiments suggest that the natural periodicityis determined by the alternation of day and night.     

Further Observations on Light and Spore Discharge in Certain Pyrenomycetes

A ‘spore-clock’ for studying the hourly rate ofspore discharge over a 24-hour period is described. A numberof the experiments reported in this paper have involved theuse of this apparatus. In Sordaria fimicola there is a distinct positive light-dischargereaction in a dark-conditioned culture, the rate of spore dischargeincreasing steeply to a peak 2–3 hours after brief stimulationby bright light. Although darkening a light-conditioned cultureleads to an immediate decrease in the rate of discharge, thereis no evidence of a delayed negative dark-discharge reaction. In S. verruculosa with a 12-hours light: 12-hours dark dailyreëgime, more spores are discharged in the dark than inthe light periods if the intensity of illumination is low. Withhigher light intensity there is no significant difference betweenthe number of spores discharged in light and dark periods. Asin S. fimicola there is a positive light-discharge reaction,the interval between stimulus and maximum response being muchlonger (8–12 hours). When a dark-conditioned culture istransferred to light for 48 hours and then returned to darknessfor a further 48 hours it is apparent that not only is therea positive light-discharge reaction but also a negative dark-dischargeresponse. The ‘plateau’ level of discharge is essentiallythe same in light and darkness. It is confirmed that in Hypoxylon fuscum light inhibits discharge.     

Microsporidian Spore Discharge and the Transfer of Polaroplast Organelle Membrane into Plasma Membrane

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.     

An Endogenous Rhythm of Spore Discharge in Sordaria fimicola

The sporulation process in Sordaria fimicola has been studiedunder controlled conditions, with particular reference to discharge.Discharge occurred with a circadian periodicity for the greaterpart of the sporulation life of the culture, with peaks at about18.00 hours in either light or darkness. The consistency ofthis phenomenon was confirmed by an analysis of previous work.Fluctuations in light intensity, temperature, relative humidity,carbon dioxide concentration, and vibration were eliminatedas far as possible, as causes of this periodicity. Minor fluctuationsin discharge rate with a period of less than 24 h were probablyrandom. The general nature of the periodicity was shown to be consistentwith some aspects of endogenous rhythms in other organisms.However, spore discharge in S. fimicola is very sensitive tolight, and it is readily entrained to discharge out of phasewith the natural period. The rate of development of two stages of spore formation (delimitationand maturation) were determined, and these were related to therate and periodicity of discharge. From the data it was impossibleto establish at which stage of sporulation the periodicity arose.     

Stimulation of Spore Discharge in Sordaria by Carbon Dioxide

The effect on the rate of spore discharge in Sordaria fimicolaof small concentrations of carbon dioxide has been studied.Substitution of an air-stream containing 0.2-2.5 per cent carbondioxide for air freed from this gas circulating over the peritheciaalways led to a marked increase in the rate of spore discharge.     

An Analysis of Spore Discharge in Sordaria

Sordaria fimicola can develop mature perithecia from which sporesare discharged when grown in darkness or in light. Under conditionsof alternating dark and light (12 hrs.: 12 hrs.) each day, sporedischarge is periodic with a low rate during the dark period,succeeded by a gradual rise to a relatively high rate in thelight period followed by a decline before the onset of the nextdark period. There is no trace of an endogenous rhythm. Transferfrom darkness to light always leads to an increase in the rateof discharge, and from light to dark to a decrease. The heightof the peak of discharge rate attained in light following adark period seems to be related to the length of the precedingdark period. Experiments with light of different colours but of roughly thesame energy value show that it is the blue rays that are mainlyeffective. From cultures of filter-paper yeast-extract mediuman orange pigment can be extracted with a maximum absorption,in the visible spectrum, at 470 mµ. It is possible thatthis is important in connexion with the sensitivity of the fungusto blue light.     

Light and Spore Discharge in Ascobolus

Spore discharge in Ascobolus crenulatus occurs both in the lightand in the dark. In a 12 h light: 12 h dark daily regime discharge-ratehas peaks in the dark periods, due apparently to light stimulationwith about half a day's interval between stimulus and response. Using a ‘spore clock’ the course of discharge hasbeen followed for a single apothecium on changing from darknessto light. Exposure to light (500 lux) of wave-lengths around400, 440 and 460 mµ immediately causes ’puffing‘,whilst light of longer wave-length (504 and 580 mµ) hasno effect. Change from darkness to white light has no immediateeffect, but there is a delayed stimulation with a marked increasein discharge-rate 10–14 h later. Simultaneous illuminationof an apothecium, which has been in darkness, by blue light(420 mµ, or 440 mµ, 500lux) and yellow light (580mµ, 500 lux) does not result in puffing. The yellow appearsto prevent the blue light from exerting its effects.     

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.     

Differential Inactivation of Fungal Spores in Water and on Seeds by Ozone and Arc Discharge Plasma

Seed sterilization is essential for preventing seed borne fungal diseases. Sterilization tools based on physical technologies have recently received much attention. However, available information is very limited in terms of efficiency, safety, and mode of action. In this study, we have examined antifungal activity of ozone and arc discharge plasma, potential tools for seed sterilization. In our results, ozone and arc discharge plasma have shown differential antifungal effects, depending on the environment associated with fungal spores (freely submerged in water or infected seeds). Ozone inactivates Fusarium fujikuroi (fungus causing rice bakanae disease) spores submerged in water more efficiently than arc discharge plasma. However, fungal spores associated with or infecting rice seeds are more effectively deactivated by arc discharge plasma. ROS generated in water by ozone may function as a powerful fungicidal factor. On the other hand, shockwave generated from arc discharge plasma may have greatly contributed to antifungal effects on fungus associated with rice seeds. In support of this notion, addition of ultrasonic wave in ozone generating water has greatly increased the efficiency of seed disinfection.     

Spore membrane(s) as the site of damage within heated Clostridium perfringens spores.

Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.     

Effects of Airspeed and Humidity Changes on Spore Discharge in Sordaria fimicola

An improved experimental system is described for studying therate of spore discharge in Sordaria fimicola subjected to anair-stream of controlled rate and relative humidity. Under nearlysaturated conditions the rate of air-flow does not seem to affectthe rate of discharge. Reduction of the R.H. from 95 per centto 70 per cent or 35 per cent leads to an immediate but temporaryincrease in discharge rate. 70 per cent R.H. seems only to affectactual discharge, but prolonged exposure to 35 per cent R.H.interferes as well with earlier stages in spore development.It is suggested that the immediate effect on discharge is physicalrather than biochemical. There is evidence that the perithecialwall plays some part in the control of spore discharge.     

枯草芽胞杆菌芽胞表面展示研究进展

枯草芽胞杆菌芽胞表面展示技术是把枯草芽胞杆菌作为芽胞表面展示的宿主来展示目的蛋白的一种技术。该技术不仅具备芽胞表面展示技术可展示分子量较大的目的蛋白、目的蛋白无需跨膜及芽胞的极强抗逆性等特点外,同时由于该技术的宿主菌--枯草芽胞杆菌的分子生物学信息研究得比较清楚、安全性高而被广泛应用。介绍了枯草芽胞杆菌表面展示近10年在生产疫苗和固定化酶方面的进展,并对如何提高表面展示目的蛋白的产量做了简要概述。     

Testing the integrity of fine spatial resolution palaeoecological records: microcharcoal data from near-duplicate peat profiles from the North York Moors, UK

Microcharcoal data which cover the millennium preceding the Ulmus Decline from near-replicate peat profiles 30 cm apart are presented from North Gill, an intensively studied upland spring-head site in north-east England with an established detailed palaeoecological record. The Ulmus Decline at this site has been consistently dated to ca. 4700 ¹⁴C years BP. The microcharcoal records from these two very closely adjacent points, which must have had virtually identical microcharcoal source areas, are assessed for their degrees of similarity and variability at 1 cm vertical sampling intervals, in an investigation which parallels a previous comparative pollen study of the two profiles. The major trends and frequency peaks and troughs of the twin microcharcoal curves were found to correspond well, suggesting that microcharcoal records from small peat mires within forested catchments have fine spatial integrity, are reproducible and can be interpreted securely as reliable records of fire history in their small source areas. Much variation occurred at the level of individual spectra within coeval zones which must record the same fire history and ecology however, producing differently shaped microcharcoal curves. While the record of major trends will be accurate, therefore, too precise an interpretation of microcharcoal data at this temporal scale may not be justified. Micro-spatial variations in frequencies of fungal spores associated with local burning are also presented and evaluated.     

Display of Human Proinsulin on the Bacillus subtilis Spore Surface for Oral Administration

With the continuous improvement of the living standards of human society, the number of diabetics worldwide is growing rapidly. To date, the main effective therapy for diabetic is intravenous injection of insulin, which is accompanied by a lot of shortage such as high cost and side effects. To obtain long-term bioactive anti-diabetic drug for oral administration, we used human proinsulin (hpi) as a foreign gene to construct a recombinant plasmid pJS700-HPI with an enterokinase site Asp-Asp-Asp-Asp-Lys suitable for digestion. Plasmid pJS700-HPI was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spores formation, western blot was used to monitor HPI surface expression on spores. Oral administration to the silkworms with spores implied that the HPI protein displayed on recombinant spores may be digested and absorbed into the silkworm’s hemolymph due to the resistant characters of spores and the addition of enterokinase site.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.